Serva Blue

Bradford reagent 50 mg Coomassie Brilliant Blue G-250 (Serva Blue G, Serva, cat. no. 35050) is dissolved in 50 ml ethanol (95%), mixed well, and added to 100 ml phosphoric acid (85%). After the dye has completely dissolved, the volume is completed to 1 1 and is filtered through Whatman 1 paper. It is stored in the dark at room temperature. 

Serva Blue W 0.8% /3-mercaptoethanol RNA loading buffer 80% formamide, v/v 0.1% xylene cyanol 0.1% bromophenol blue 1 mM EDTA, pH 8.0... 

Problems Serva Blue G presumably prefers to attach to trans-membrane regions. Large membrane proteins thus show a lesser charge density than small membrane proteins. Soluble proteins bind still less stain. The charge-to-mass ratio of different protein-stain complexes is thus not constant, and the native electrophoresis does not separate the protein complexes by MW (personal communication by A. Schrattenholz, Mainz). Soluble marker proteins such as thyroglobulin, ferritin, and the like smudge in the gel or partially disintegrate into subimits. 

Serva Blue G binds tightly to nitrocellulose and PVDF membranes. The blot of a blue gel is thus blue, and this interferes with the immunostain or ligand coloring of the blot (see Section 1.6.3). Finally, the run of a blue gel lasts 3 to 6 h, and the bands are blurry. 

B. Bradford assay (Bradford 1976) Coomassie brilliant blue G-250 (Serva Blue G, Serva). The reagent for this assay is commercially available from Bio-Rad (Cat. No. 500-0006). 

Protein reagent stock solution 0.05% (w/v) Coomassie brilliant blue G-250, 23.8% (v/v) ethanol, 42.5% (w/v) phosphoric acid. To make 200 ml of stock solution (5000 determinations), dissolve 0.1 g Serva blue G in 50 ml 95% ethanol (denatured ethanol usually works as well), add 100 ml 85% phosphoric acid, and make up to 200 ml by adding water. The stock solution is commercially available (Bio-Rad). Keep at 4°C. 

Figure 3 shows one-dimensional gels of histones (Figs. 3A and B) and nonhistone proteins (Fig. 3C) run in a 15% acrylamide Laemmli gel and stained with Coomassie brilliant blue. Representative silver-stained gels of 16.5% T, 6% of proteins from three different cell types are shown in Fig. 4. Serva blue G migrates as a broad band in front of the smallest proteins in the gels as described here. This is not always the case for the commonly used bromophenol blue.

Coomassie Brilliant Blue G-250 (color index 42655) is available from a number of different suppliers (e.g., ACROS, Aldrich, AMRESCO, Bio-Rad, Fisher Biotech, Fluka, ICN Biomedicals, J.T. Baker, Research Organics, Serva, Sigma, or USB). 

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